1. Field of the Invention
The present invention relates to an apparatus and method using enzyme electrodes for simultaneously measuring the concentration of two substrates such as glucose and sucrose in a liquid, and more particularly to improvements in the measuring apparatus and method wherein the same enzymatic reaction for forming a detectable substance occurs in each of a pair of electrodes, and the substance to be finally detected by each electrode is identical.
2. Description of the Prior Art
Enzyme electrodes are excellent in readiness and promptness, and have come to be used widely in various fields including clinical analysis, food analysis and environmental analysis.
Recently, in particular, great efforts are concentrated in the development of simultaneously measuring apparatus capable of measuring the concentration of two different substances using enzyme electrodes, and such simultaneous measuring apparatus for measuring, for example, glucose and uric acid (see Japanese Laid-open Patent Application No. 62-24142), lactic acid and pyruvic acid (see Japanese Laid-open Patent Application No. 62-5172), and others have been introduced, in which each of the employed enzymes function as the catalyst for a reaction with a specific object of measurement.
An apparatus for measuring the concentration of two substrates simultaneously was also disclosed by the present inventors, wherein the same enzymatic reaction for forming or consuming a detectable substance occurs in each of a pair of electrode, and the substance to be finally detected by each electrode is identical (see Japanese Laid-open Patent Application No. 64-69944, EP0310824).
A measuring method of sucrose is disclosed, in which the sucrose is hydrolyzed by invertase into fructose and .alpha.-D-glucose, and .alpha.-D-glucose is converted into .beta.-D-glucose by mutarotase, and then .beta.-D-glucose is oxidized by glucose oxidase to produce an electrode active substance of hydrogen peroxide, which is detected electrochemically (C. Bertrand, P. R. Coulet, D. C. Gautheron, Anal. Chim. Acta, 126, 23-34, 1981). On the other hand, to measure the concentration of glucose, the glucose is oxidized by glucose oxidase, and the produced hydrogen peroxide is electrochemically detected. That is, both in the measurement of sucrose and the measurement of glucose, the oxidation of glucose is a common reaction to produce an electrode active substance, and the substance detected by the electrode is hydrogen peroxide.
Therefore, the present inventors disclosed the method to measure simultaneously glucose and sucrose by using enzyme electrodes. The concentration of glucose is determined from the output voltage detected by the enzyme electrode having immobilized glucose oxidase. To measure the sucrose concentration, on the other hand, the enzyme electrode for detecting both glucose and sucrose having immobilized glucose oxidase, mutarotase and invertase is set in a flow type measuring apparatus. By using the previously detected concentration of glucose, the contribution portion of the glucose initially contained in the sample is calculated from the preliminarily calibrated value, concerning the enzyme electrode having immobilized glucose oxidase, mutarotase and invertase, and this value is subtracted from the output current value to determine the sucrose concentration.
That is, generally, the concentrations of two components are measured by the following methods.